Chromatographic purification and separation process for mixtures of nucleic acids

ABSTRACT

A method for the purification and separation of nucleic acid mixtures by chromatography including adsorbing the nucleic acids to be separated and purified from a solution with a high concentration of salts (ionic strength) and/or a high concentration of alcohol on a substrate and subsequent desorbing from the substrate by means of a solution with lower concentration of salts (ionic strength).

The object of the present invention is a method for the purification andseparation of nucleic acid mixtures by chromatography, the use of saidmethod for purifying nucleic acid fragments that have been subjected tomodification reactions, a device for performing said method, an aqueoussolution which can be used in the method according to the invention, andthe use of said solution.

Adsorbing nucleic acids on glass or silica-gel particles in the presenceof chaotropic salts is well-known (Vogelstein, B. and Gillespie,D.(1979); Preparative and analytical purification of DNA from agarose,Proc. Natl. Acad. Sci. USA 76: 615-619). According to this method, usinghigh concentrations of chaotropic salts, such as sodium iodide, sodiumperchlorate, or guanidine thiocyanate, DNA is isolated and purified fromagarose gels and RNA and DNA preparations are isolated and purified fromvarious extracts (Boom, R. et al. (1990); Rapid and simple method forpurification of nucleic acids, J. Clin. Microbiol. 28, 495-503, andYamado, O. et al. (1990); A new method for extracting DNA or RNA forpolymerase chain reaction, J. Virol. Methods 27, 203-210). Although thephysical processes resulting in an adsorption of the nucleic acids onmineral substrates in the presence of chaotropic reagents are notunderstood in detail, it is believed that the reason of this adsorptiondwells in disturbances of higher-order structures of the aqueous medium.This leads to adsorption or denaturation of the dissolved nucleic acidon the surface of the glass or silica-gel particles. In the presence ofhigh concentrations of chaotropic salts, this adsorption will occuralmost quantitatively. Elution of the adsorbed nucleic acids isperformed in the presence of buffers having low ionic strengths (saltconcentrations). The prior art methods allow for the treatment ofnucleic acids and fragments ranging in size from 100 base pairs (bp) to50,000 base pairs (bp). To date, it has not been possible, however, toquantitatively separate short single-stranded or double-stranded nucleicacid fragments (100 bp and smaller) from very short (20 to 40nucleotides) single-stranded oligonucleotides (e.g. primers).

Typically, such nucleic acid mixtures are formed as amplificationproducts, for instance by polymerase chain reaction (PCR). In manycases, the products resulting from this reaction are subsequentlyanalyzed in terms of molecular biology, using the conventionaltechniques such as DNA sequencing, DNA hybridization, cloning,restriction enzyme analysis, and transformation. Thereby, analyticalparameters, such as information about genetic mutations for geneticadvising or detection of pathogens in medical diagnostics (e.g. HIV),can be obtained. To be capable of making full use of the potential ofthose diagnostic methods, quantitative separation or purification ofthese DNA fragments which are often quite small (100 base pairs) is veryimportant.

Presently available purification methods are based on ultrafiltration,high pressure liquid chromatography (HPLC), or extraction of nucleicacid fragments from agarose gels in the presence of chaotropic salts byprecipitaion onto glass or silica-gel particles. For the separation ofnucleic acid mixtures comprising for example a double-stranded DNAfragment (100 bp) and a smaller single-stranded oligonucleotide (forinstance 39-mer), however, these methods are useful only with lowefficiency.

The technical problem of the present invention consists in providing amethod allowing to avoid the above-mentioned drawbacks of the prior art.This problem is solved by a method for the purification and separationof nucleic acid mixtures by chromatography according to the featuresdescribed herein.

An embodiment of the invention pertains to the use of the methodaccording to the invention for the purification of nucleic acidfragments following modification reactions, a device for performing themethod according to the invention, and an aqueous solution that can beused in the method according to the invention, and a combination of thedevice and the aqueous solution.

The method according to the invention makes use of the per se knownproperty of nucleic acids to precipitate onto mineral substrates in thepresence of chaotropic salts, solutions of salts having high ionicstrengths (high concentrations), reagents such as e.g. urea, or mixturesof such substances and to be eluted by the action of solutions of lowionic strengths (salt concentrations). Thus, Applicant's PCT/EP 92/02775suggests to first adsorb a nucleic acid mixture contained in a medium oflow ionic strength on an anion-exchanging material, to subsequentlydesorb the nucleic acid by means of a buffer of higher ionic strengthand then to adsorb the nucleic acids contained in the buffer of thishigher ionic strength on a mineral substrate material in the presence oflower alcohols and/or polyethylene glycol and/or organic acids, such astrichloroacetic acid (TCA). Thereafter, the nucleic acids are elutedpreferably by means of water or a buffer solution of low ionic strength.

It has now been found that for the separation of nucleic acids,preliminary purification on anion-exchanging materials can be dispensedwith. Surprisingly, excellent fractioning of a nucleic acid mixture canalso be accomplished by adsorbing the nucleic acids in the presence ofhigh concentrations of chaotropic salts and desorbing the nucleic acidsby means of solutions of low ionic strengths.

Thus, the method according to the invention allows for efficientlyobtaining nucleic acid fractions of interest in one operation stepwithout preliminary purification steps by adsorbing the nucleic acids tobe separated and eluting them.

If samples containing nucleic acids are to serve as sources of thenucleic acids to be purified and isolated, those sources are digested ina per se known manner, for example by treatment with detergents or bymechanical action, such as ultrasonic waves or disintegration. Thesolution used to receive the nucleic acids may already contain highconcentrations of chaotropic salts. After larger cell constituents thatmay be present have been removed by centrifugation or filtration (WO93/11218 and WO 93/11211), the solution is contacted with a mineralsubstrate material in order to adsorb the nucleic acids from thesolution having high ionic strength of chaotropic salts on the mineralsubstrate.

A modification of the method according to the invention consists inperforming digestion of the nucleic acids directly within the buffersystem employed for the adsorption. In this case, a particularlyfavorable nucleic acid distribution can be obtained.

Nucleic acids are commonly obtained from eukaryotic and/or prokaryoticcells (including protozoans and fungi) and/or from viruses. For example,the cells and/or viruses are digested under highly denaturing and, ifappropriate, reducing conditions (Maniatis, T., Fritsch, E. F., andSambrook, S., 1982, Molecular Cloning Laboratory Manual, Cold SpringHarbor University Press, Cold Spring Harbor).

One particular embodiment of the invention is especially useful for theisolation of plasmid or cosmid DNA from E. coli. Following lysis of theE. coli cells with sodium hydroxide/SDS, the solution is neutralizedwith potassium acetate (KAc, 0.2-0.9 M).

Normally, the cell lysate is neutralized after SDS lysis by means of 3 Mpotassium acetate. Then, in order to centrifuge off the cell fragments,5 M guanidine hydrochloride or another high concentration solution ofchaotropic salt is added to the cell lysate. With E. coliminipreparations, this will yield about 2-3 ml of the sample to beadsorbed on silica gel which is unfavorable, however, since it will haveto be centrifuged off then which takes several hours.

After lysis with sodium hydroxide/SDS, the method according to theinvention uses e.g. solutions of salts preferably containing

0.2 M KAc / 5.5 M GuHCl, 0.2 M KAc / 5.5 M GITC, 0.2 M NaAc / 6 MNaClO_(4,) 0.2 M NaAc / 6 M GuHCl, or 0.2 M NH₄Ac / 6 M NaClO₄.

Thereby, neutralization of the cell lysate and concurrent adjustment ofthe sample to high salt concentration conditions in silica gel isachieved resulting in substantial facilitation of work in everydaypractice. Surprisingly, it has further been found that adsorption ofnucleic acids on silica gel will also take place in the presence ofanionic or cationic or neutral detergents, such as e.g. SDS, NP40, Tween20, Triton X-100, CTAB, in combination with chaotropic salts, or thatthe presence of these detergents will even increase the DNA yield.

Widely used is the lysis of cells by means of detergents as denaturingreagents and degradation by particular enzymes of the protein structuresand nucleic acid cleaving enzymes. Thus, sodium dodecylsulfate (SDS) andEDTA, for instance, are used as denaturing agents and proteinase K isused to degrade proteins. In most cases, the result of such lysingprocedure is a highly viscous jelly-like structure from which thenucleic acids are isolated by phenol extraction, wherein long portionsof the nucleic acids remain intact. After dialysis and precipitation,the nucleic acids are removed from the aqueous phase. This lysingprocedure is such aggressive towards non-nucleic acid structures thatpieces of tissue may also be subjected to it.

Due to this labor-intensive technique involving repeated change ofreaction vessels, however, this method is unfavorable with large amountsof samples and routine preparations. Although this method can beautomated, a commercially available device of this kind presently willmanage about 8 samples at a time within four hours (Applied Biosystems A371). Thus, this method is expensive and unsuitable for passing largeseries of samples. Another drawback is that subsequent reactions such asenzymatic amplification are adversely affected due to the large lengthsof the isolated nucleic acids. In addition, the solutions obtained arehighly viscous and difficult to handle. In particular, DNA of very largelength rather is obtrusive since nucleic acids obtained by the prior artmethod have to be cleaved in a separate step to be further processed.

Although digestion of eukaryotic and/or prokaryotic cells and/or virusesin alkaline medium in the presence of detergents is technically simple,it also yields nucleic acids of large lengths which are unfavorable asdescribed above.

The rough preparation of the nucleic acids is followed by subsequentreactions. Those subsequent reactions require a certain quality ofnucleic acids. For instance, said nucleic acids must be largely intact,the yield of the preparation must be high and reproducible, and inaddition, the nucleic acids must be present in high purity, devoid ofproteins and cellular metabolites. The preparation route must be simpleand economic and allow for automation. The preparation of the nucleicacids must be possible without the risk of cross-contamination withother samples, especially when enzymatic amplification reactions areused, such as polymerase chain reaction (PCR) (Saiki, R., Gelfand, D.H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B.,and Ehrlich, H. A. (1988), Science 239, 487-491) and ligase chainreaction (LCR) (EP-A-88 311 741.8). For those subsequent reactions, itis desirable to obtain the nucleic acids in not too large chain lengths,to lyse the cells quantitatively, if possible, and in addition to avoidthe above-mentioned drawbacks of the digestion methods known in theprior art.

Hence, it is desirable that a method allow for the isolation andconcentration of nucleic acids from intact eukaryotic and/or prokaryoticcells and/or viruses or from body fluids. In particular, the nucleicacid thus obtained should be characterized by not too large chainlengths, be isolatable in a few steps and be capable of being directlysubjected to the required subsequent reactions.

The modification, set forth above, of the method according to theinvention allowing for this consists in lysing the sources of thenucleic acids, such as eukaryotic and/or prokaryotic cells and/orviruses.

Said digestion of nucleic acid containing sources, such as eukaryoticand/or prokaryotic cells and/or viruses, may preferably be performed byphysical or chemical action. Lysis may be accomplished eithermechanically, such as by ultrasonic waves or by osmotic shock, orchemically by means of detergents and/or chaotropic agents and/ororganic solvents (e.g. phenol, chloroform, ether) or by alkalinedigestion.

This procedure results in the preparation of nucleic acids with highpurity and allows to perform qualitatively and quantitativelyreproducible analytics, especially in combination with enzymatic methodsfor the amplification of nucleic acids. Digestion methods usingdetergents and/or chaotropic agents, concentrated solutions of salts,reagents such as urea, mixtures of these substances, and/or organicsolvents or physical digestion methods such as heating of a sample haveproven to facilitate subsequent applications. For instance, when themethod according to the invention is used, shorter cellular DNA (<50 kb)or total nucleic acids from cells and/or viruses and/or body fluids areobtained. The purification method (i.e. the conditions while the nucleicacids are bound and eluted) results in fragmentation of the nucleicacids.

The combination of chaotropic agents with high ionic strengths andhydrophobic organic or inorganic polymers and/or alcohols and/ortrichloroacetic acid (TCA) in the adsorption buffer ensures that incontrast to conventional purification methods the nucleic acids arequantitatively fixed with high specifity on the surface of the mineralsubstrate material, such as quartz fibers, following lysis and thus areprotected from further nuclease attacking while contaminating componentsof the lysate will not bind. In this state of the nucleic acids beingfixed, residual contaminating components are readily washed out, withsubsequent elution of the pure nucleic acid in a smaller volume. Thus,reproducible average chain lengths of 20 to 40 kb are obtained. Underthe conditions of digestion as described in examples 7 to 9, less than10% are shorter than 10 kb. This represents an optimum lengthdistribution for subsequent enzymatic nucleic acid amplification.

The special combination of salts, particularly chaotropic agents, andalcohols for the first time allows for concurrently isolating andpurifying nucleic acids of a broad spectrum of chain lengths (10-100,000base pairs).

The aqueous adsorption solution with a high concentration of saltscontains 1 to 50% by volume of an aliphatic alcohol with a chain lengthof from 1 to 5 carbon atoms or polyethylene glycol.

Suitable mineral substrates are porous or non-porous materials based onmetal oxides and mixed metal oxides, such as those made of silica gel,materials principally consisting of glass, alumina, zeolites, titaniumdioxide, zirconium dioxide. Zeolites in particular have proven to besuitable mineral substrates.

Optionally, the mineral substrate material having the nucleic acidsadsorbed thereon may be washed with a solution which, due to arelatively high alcohol content, will prevent the nucleic acids frombeing desorbed.

Then, the adsorbed nucleic acids are eluted with a buffer of low saltconcentration (ionic strength), and the nucleic acids or nucleic acidfractions obtained are collected.

Suitable chaotropic salts are sodium perchlorate, guanidinehydrochloride (GuHCl), guanidine isothiocyanate (GTC), potassium iodidein concentrations of from 1 to 8 M. Also useful are concentratedsolutions of salts, >1 M NaCl, KCl, LiCl, etc., reagents such as urea(>1 M), and combinations of such components. The lower alcohols presentin the solution of the chaotropic salts are methanol, ethanol,isopropanol, butanol, and pentanol in amounts of 1 to 50%, inasmuch asthey are miscible with water within these ranges. The ethylene glycolswhich may be preferably used have molecular weights of from 1,000 to100,000, particularly of from 6,000 to 8,000. Said polyethylene glycolmay be added to the buffer having high ionic strength in amounts of from1 to 30%.

The particle size of the mineral substrate materials preferably is from0.1 μm to 1,000 μm. If porous mineral substrates, such as for instanceporous silica gel, porous glass, porous alumina, zeolites, are used, thepore sizes preferably are from 2 to 1,000 nm. The substrate material canbe present, for instance, in the form of loose fillings and be contactedwith the solutions containing nucleic acids to be separated andpurified.

Preferably, however, the porous and non-porous substrate materials arein the form of filter layers arranged in some hollow body provided withan inlet and an outlet. The filter layers either consist of directed(woven) or undirected fibers made of glass, quartz, ceramics, or othermaterials, such as minerals, or they consist of a membrane in whichsilica gel is incorporated.

The method according to the invention is excellently useful for theseparation of nucleic acid mixtures, including in particular short-chainnucleic acids having only slightly different chain lengths. Thus, DNAfragments with a size of 100 bp, for example, can be separated fromsmaller single-stranded oligonucleotides, for instance a 39-mer. In thiscase, the yield in DNA is then increased by 60 to 70% as compared withother conventional purification methods, such as ultrafiltration, HPLC,or the use of chaotropic salts alone.

When the method according to the invention is employed in which thedigestion of the sources containing nucleic acids is performed in thereceptive (adsorption) buffer, preparation of nucleic acids with adefinite nucleic acid length spectrum is possible.

The method according to the invention allows for processing nucleic acidmixtures of every origin whatever. Thus, nucleic acids from biologicalsources such as all kinds of tissues, body fluids, such as blood, fecalmatter after appropriate sample priming, which at any rate comprisesincorporation of the sample in a solution with a high concentration ofsalts, preferably a high concentration of chaotropic ions, can beobtained. Nucleic acids formed by chemical reactions, such as thoseobtained by polymerase chain reaction (PCR), or plasmid DNA, genomic DNAand RNA and/or nucleic acids derived from microorganisms can also beseparated and purified according to the invention.

The method according to the invention may also include the use ofso-called plasmid DNA minipreparations from Escherichia coli forsubsequent cloning or sequencing; the method according to the inventionis also useful for isolating DNA and/or RNA from whole blood, plasma,serum, tissues, cell cultures, bacteria, in particular Mycobacteriumtuberculosis, viruses, such as cytomegalovirus (nucleic acid DNA), RNAviruses, such as HIV, hepatitis B, hepatitis C, hepatitis δ viruses.Oligonucleotides are also nucleic acids within the meaning of the methodaccording to the invention. Furthermore, the nucleic acids may bederived from sequencing reactions or other comparable reactions.Preparation of DNA or RNA from whole blood is particularly useful forsubsequent determination of HLA type. The method according to theinvention is particularly useful for isolating nucleic acids fromMycobacterium tuberculosis. This involves the necessity of ratherdrastic digesting methods, with conventional isolation techniquesyielding only unsatisfactory results.

A device which may be preferably used in the method according to theinvention is a hollow body, especially of cylindrical shape, providedwith an inlet and an outlet. In the vicinity of the outlet, seen in thedirection of flow of the solution through the hollow body, the mineralsubstrate material on which the nucleic acids are to be adsorbed islocated. A means which in a preferred embodiment consists of twopolyethylene frits arranged one above the other leaving some spacebetween them fixes the substrate material, which is located in saidspace between the polyethylene frits, within the lumen of the hollowbody. The means for fixing the substrate material may also be aself-supporting membrane in which the substrate material is embedded.Attachment of the substrate material or of the means fixing thesubstrate material can be effected by frictional or tensional forcesgenerated for instance by clamping said means within the hollow bodyand/or by fixing said means with a tension ring.

The pore size of said means, preferably polyethylene or polypropylenefrits, must be large enough to allow the lysate components to passthrough without obstruction. Preferably said means have pore sizes from5 to 200 μm. This device for the first time allows for simple, rapid andreproducible isolation of nucleic acids even from highly viscous lysateswith a very high protein content (e.g. blood lysates which have a veryhigh content of hemoglobin).

In an especially preferred embodiment, the mineral substrate material isa reticular membrane made of silica-gel, glass or quartz fibers havingpore sizes of <5 μm on which the liberated nucleic acids are adsorbed.

Another preferred embodiment is represented by a device in which themineral substrate material is a particular inorganic polymer such assilica gel or quartz gel with particle sizes of from 1 to 50 μM.

Said hollow body may be a commercially available tube, for instance.Between the two means being tightly pressed in, for instancepolyethylene frits having pore sizes of 50 to 200 μm, there is one ormore layers of a membrane having pores with sizes ranging from 0.1 to 1μm which membrane is made of silica, glass or quartz fibers or of silicagels. This membrane has a thickness of about 0.2 to 1.0 mm, especiallyof 0.6 mm.

The capacity of the membrane material is about 20 to 100 μg of DNA. Ofcourse, by stacking such membranes on top of one another, the capacityfor DNA may be increased. When there is only small mechanical strain,welding or sticking of the membrane edges to the device may also beconsidered in which case the stabilizing effect of said means may bedispensed with, such that the membrane will seal the hollow body withoutsaid means. The membrane may then be fixed within the hollow body byplacing a tension ring.

It is also possible to fill small columns with the silica gel describedbeing located between 2 polyethylene frits having pore sizes of 35 μm.Preferably, the top frit is selected to have larger pores (10-250 μm,especially 50 μm). Said columns are preferably charged with about 70 mgof silica gel corresponding to a filling level of 3 mm.

Also preferred is the use of the above-mentioned method in strips with 8parallel preparation facilities each, in the microtiter plate format (96facilities for almost simultaneous preparation), and/or in combinationwith a filtration step and/or desalting step (see Applicant's PatentApplications P 41 27276.5, P 41 39 664.2).

In a preferred embodiment of the device, a polyethylene frit with athickness of 0.5 to 1.5 mm and pore sizes of about 10 μm is clamped intoa centrifuge chromatographic column in the shape of an essentiallycylindrical hollow body. On this frit, there is charged a layer, abouttwice as thick, of silica gel having particle sizes of about 10 to 15 μmand pore sizes of 40 to 120 Å which is sealed by a second frit that maybe of the same kind as the first frit. Preferably, the silica-gel layermay be condensed by pressure between the frits.

Another embodiment of the chromatographic column comprises glass fiberfragments having lengths of 10 to 300 μm as a substrate material locatedbetween two polyethylene frits with pore sizes of about 50 μm. Othersuitable substrate materials are glass fiber papers, quartz fiberpapers, glass fiber fabrics and other mineral papers and fabrics.

Another preferred embodiment of the device comprises a membrane in thevicinity of the outlet having silica-gel particles embedded therein. Inthis case, the membrane which preferably is self-supporting especiallymay be fixed by means of a tension ring. As a silica-gel membrane, anEmpore silica-gel membrane of the firm of 3M may be used to advantage. Asilica-gel membrane consisting of silica gel and porous PVC may also befixed within the lumen of the cylindrical hollow body, especially bymeans of a tension ring.

In a preferred embodiment of the method according to the invention, thedescribed device in one of its embodiments, for example, is charged withthe solution of the nucleic acid mixture to be separated. Then, thesolution is passed through the mineral substrate by suction orcentrifugation or some equivalent measure as well as combinationsthereof. The nucleic acids are then adsorbed on the substrate materialas long as the solution has high ionic strength (salt concentration).

The invention will be illustrated in more detail by means of thefollowing examples.

EXAMPLE 1

Isolation of High Copy Plasmid DNA

E. coli cells from a 3 ml HB 101 culture and containing the pUC 18plasmid are centrifuged off and resuspended in 0.25 ml of buffer P1 (10mM Tris-HCl, pH 8, 100 μg/ml RNase A) and lysed by adding 0.25 ml ofbuffer P2 (0.2 M NaOH, 2% SDS). The sample is neutralized by adding 0.35ml of buffer N3 (4.2 M guanidine hydrochloride, 0.9 M potassium acetate,pH 4.8) and is at the same time adjusted to a final concentration of1.75 M GuHCl. This concentration ensures binding without requiringfurther steps. The lysed sample is centrifuged for 10 min in anEppendorf minicentrifuge at 13,000 rpm in order to remove cell fragmentsand the precipitated SDS. The supernatant containing the plasmid DNA isimmediately pipetted onto a centrifuge chromatographic column. Thiscentrifuge chromatographic column is centrifuged in a 2 ml centrifugetube and washed by renewed centrifugation of 0.5 ml of PB buffer (5 Mguanidine hydrochloride, 30% isopropanol), in order to remove impuritiesand proteins. The centrifuge chromatographic column is washed salt-freeonce by centrifuging through 80% ethanol/water and subsequently iscentrifuged for 30 to 60 sec to completely remove excess ethanol. Forelution, 0.05-0.2 ml of elution buffer (10 mM tris/HCl, pH 8.5) arecentrifuged through the centrifuge chromatographic column into a 1.5 mlcentrifuge tube. The plasmid DNA is then present in concentrated form ina solution with a very low concentration of salts. The yield is 15 μg to20 μg of plasmid DNA with an A260/A280 ratio of 1.75.

EXAMPLE 2

Isolation of High Copy Plasmid DNA from 5 ml Cultures

According to example 1, a cell lysate of a 5 ml pUC 18 plasmid/XL 1 Blueculture is prepared and centrifuged through a centrifuge chromatographiccolumn containing a silica-gel filling and washed. The plasmid DNA iseluted with 0.1 ml of TE buffer (10 mM tris/HCl, pH 8.5, 1 mM EDTA)heated at 80° C. The yield is 15-20 μg of plasmid DNA with an A260/A280ratio of 1.7.

EXAMPLE 3

Isolation of Low Copy Plasmid DNA

According to example 1, a cell lysate of a 5 ml pBR322 plasmid/XL 1 Blueculture is prepared and centrifuged through a centrifuge chromatographiccolumn containing a glass or quartz fiber membrane and washed. Theplasmid DNA is eluted with 0.1 ml of TE buffer (10 mM tris/HCl, pH 8.5,1 mM EDTA) heated at 80° C. The yield is 5-10 μg of plasmid DNA with anA260/A280 ratio of 1.7.

EXAMPLE 4

Purification of Amplification Products

A 100 μl PCR amplification reaction is mixed with 500 μl of PB buffer (5M GuHCl, 30% isopropanol); preliminary separation of the paraffin oillayer covering the reaction mixture is not necessary. This mixture ispipetted onto a centrifuge chromatographic column containing asilica-gel membrane and centrifuged in a 1.5 ml centrifuge tube. Thecentrifuge chromatographic column is washed almost salt-free bytreatment with 80% EtOH/water. For elution, 50 μl of elution buffer (10mM tris, pH 8.5) are centrifuged through the centrifuge chromatographiccolumn into another centrifuge tube. The PCR product thus purified isfree from primers, dNTPs, polymerase, and salts and can be employed, forexample, directly in a sequencing reaction in an ABI sequencer using the“Cycle Sequencing” protocol.

EXAMPLE 5

Purification of DNA Following Restriction Reactions

1 μg of DNA is treated with a restriction endonuclease. This DNArestriction reaction is mixed with 500 μl of PB buffer according toexample 4, and further processing is as in example 4. The DNA obtainedafter elution is free from restriction endonucleases and salts, and the260/280 ratio is 1.8.

EXAMPLE 6

Purification of DNA Following Enzymatic Radioactive Labeling

1 μg of DNA are radiolabeled in presence of γ-³²P-ATP by means ofoligolabeling procedure. The reaction mixture is treated as in example4. Thereby, the labeled DNA is purified from non-incorporated dNTPs,γ-³²P-ATP, salts and Klenow polymerase and may directly be employed forhybridization reaction.

EXAMPLE 7

Preparation of Nucleic Acids from Blood

Total nucleic acids preparation from blood: To 200 μl of citrate,heparin or EDTA blood in a 1.5 ml PPN tube are added 200 μl of a 4-8 Msolution of a chaotropic salt (guanidine hydrochloride (GuHCl),guanidine isothiocyanate (GTC), potassium iodide), optionally an organicsolvent (phenol, chloroform, ether), and a 5-100% detergent (NP40; Tween20, Triton X-100, SDS, CTAB). Then, 200-1000 μg of a protease is added,and the mixture is incubated for 10 min at 70° C. or for a longer periodof time at lower temperatures (e.g. for 30 min at room temperature). Inthis step, efficient lysis of all eukaryotic and/or prokaryotic cellsand/or viruses (with concomitant inactivation of infective pathogens)and denaturing and enzymatic degrading of proteins (with concomitantremoval of the proteins bound to the nucleic acids) are taking placesimultaneously. Adding 210 μl of a 95-100% alcohol (methanol, ethanol,n-propanol, isopropanol, PEG, secondary and tertiary, short-chain orlong-chain alcohols) provides highly specific binding conditions fornucleic acids, and the lysate thus adjusted is transferred to thedevice. Then, the lysate is passed through the membrane or gel matrix bycentrifuging or applying pressure, with reversible binding of thenucleic acids to the membrane fibers or gel particles. Impurities, suchas proteins, heme, heparin, iron ions, metabolites, etc., are washed outwith 0.7 ml of 100 mM NaCl, 10 mM tris/HCl, pH 7.5, 30-80% of a purealcohol (methanol, ethanol, n-propanol, isopropanol, PEG, secondary andtertiary, short-chain or long-chain alcohols) or a mixture of alcohols.DNA is eluted either with a buffer with low concentration of salts (10mM tris/HCl, pH 9.0) or with destined (deionized) water. The advantageof such elution procedures is that the DNA thus obtained may be directlyused in subsequent reactions, especially PCR, without furtherprecipitation or buffer-exchange steps. Preparation of nucleic acidsfrom other body fluids, such as e.g. semen, sputum, urine, feces, sweat,saliva, nasal mucus, serum, plasma, cerebrospinal fluid, etc., is alsopossible.

This simple method for the isolation of nucleic acids possesses a largepotential for automation, especially in combination with the subjectmatters of Applications DE-A 41 27 276.5, WO 93/11218, WO 93/11211, andDE-A 41 39 664.2.

EXAMPLE 8

Total Nucleic Acids Preparation from Extremely Small Amounts or Tracesof Blood

To 1-50 μl of citrate, heparin or EDTA blood, or of frozen and rethawedblood, or of blood renatured from dried traces in textile tissuescontained in a 1.5 ml PPN tube are added 1-50 μl of a 4-8 M solution ofa chaotropic salt (guanidine hydrochloride, guanidine isothiocyanate,potassium iodide), optionally an organic solvent (phenol, chloroform,ether), and a 1-1000 detergent (NP40; Tween 20, Triton X-100, SDS,CTAB). Then, 1-200 μg of a protease is added, and the mixture isincubated for 1 min at 70° C. or for a longer period of time at lowertemperatures (e.g. for 10 min at room temperature).

In this step, efficient lysis of all eukaryotic and/or prokaryotic cellsand/or viruses (with concomitant inactivation of infective pathogens)and denaturing and enzymatic degrading of proteins (with concomitantremoval of the proteins bound to the nucleic acids) are taking placesimultaneously. Adding ½ of volume of a 95-100% alcohol (methanol,ethanol, n-propanol, isopropanol, secondary and tertiary, short-chain orlong-chain alcohols) or of organic polymers (PEG) provides highlyspecific binding conditions for nucleic acids, and the lysate thusadjusted is transferred to the device. Then, the lysate is passedthrough the membrane or gel matrix by centrifuging or applying pressure,with reversible binding of the nucleic acids to the membrane fibers orgel particles. Impurities, such as proteins, heme, heparin, iron ions,metabolites, etc., are washed out with 0.7 ml of 100 mM NaCl, 10 mMtris/HCl, pH 7.5, 30-80% of a pure alcohol (methanol, ethanol,n-propanol, isopropanol, PEG, secondary and tertiary, short-chain orlong-chain alcohols) or a mixture of alcohols. DNA is eluted either witha buffer with low concentration of salts (10 mM tris/HCl, pH 9.0) orwith destilled (deionized) water. The advantage of such elutionprocedures is that the DNA thus obtained may be directly used insubsequent reactions, especially PCR, without further precipitation orbuffer-exchange steps.

This method is also useful for the preparation of nucleic acids fromextremely small amounts of other body fluids (semen, sputum, urine,feces, sweat, saliva, nasal mucus, serum, plasma, cerebrospinal fluid,etc.) or dried traces thereof.

This simple method for the isolation of nucleic acids possesses a largepotential for automation, especially in combination with the subjectmatters of Applications DE-A 41 27 276.5, WO 93/11218, WO 93/11211, andDE-A 41 39 664.2.

EXAMPLE 9

Total Nucleic Acids Preparation from Tissues

To 100 μg to 10 mg of a tissue in a PPN tube are added a 4-8 M solutionof a chaotropic salt (guanidine hydrochloride, guanidine isothiocyanate,potassium iodide), optionally an organic solvent (phenol, chloroform,ether), and a 5-100% detergent (NP40; Tween 20, Triton X-100, SDS,CTAB), and the whole is homogenized by means of a commercially availablehomogenizer or by mortarpounding in liquid nitrogen. Then, 100-1000 μgof a protease is added, and the mixture is incubated for 10-20 min at70° C. or for a longer period of time at lower temperatures (e.g. for30-60 min at room temperature). In this step, efficient lysis of alleukaryotic and/or prokaryotic cells and/or viruses (with concomitantinactivation of infective pathogens) and denaturing and enzymaticdegrading of proteins (with concomitant removal of the proteins bound tothe nucleic acids) are taking place simultaneously. Adding ½ of volumeof a 95-100% alcohol (methanol, ethanol, n-propanol, isopropanol, PEG,secondary and tertiary, short-chain or long-chain alcohols) provideshighly specific binding conditions for nucleic acids, and the lysatethus adjusted is transferred to the device. Then, the lysate is passedthrough the membrane or gel matrix by centrifuging or applying pressure,with reversible binding of the nucleic acids to the membrane fibers orgel particles. Impurities, such as proteins, heme, heparin, iron ions,metabolites, etc., are washed out with 0.7 ml of 100 mM NaCl, 10 mMtris/HCl, pH 7.5, 30-80% of a pure alcohol (methanol, ethanol,n-propanol, isopropanol, PEG, secondary and tertiary, short-chain orlong-chain alcohols) or a mixture of alcohols. DNA is eluted either witha buffer with low concentration of salts (10 mM tris/HCl, pH 9.0) orwith destilled (deionized) water. The advantage of such elutionprocedures is that the DNA thus obtained may be directly used insubsequent reactions, especially PCR, without further precipitation orbuffer-exchange steps.

This simple method for the isolation of nucleic acids will functionreproducibly from all tissues including tumors, inter alia, andpossesses a large potential for automation, especially in combinationwith the subject matters of Applications DE-A 41 27 276.5, WO 93/11218,WO 93/11211, and DE-A 41 39 664.2 of the same applicant.

EXAMPLE 10

Purification of DNA Fragments from Agarose Gels

A DNA fragment is separated in an agarose gel (TAE or TBE 0.5-2%). TheDNA fragment to be isolated is cut out of the gel and mixed with 300 μlof QX1 buffer (7 M NaPO₄, 10 mM NaAc, pH 5.3) in a 1.5 ml Eppendorfvessel. After incubation for 10 minutes at 50° C., the agarose will havedissolved. This solution is placed on a centrifuge chromatographiccolumn according to example 1 and centrifuged through. Then, thecentrifuge chromatographic column is washed salt-free by centrifuging of80% ethanol/water through the column and subsequently is centrifuged for1 min to completely remove excess ethanol. For elution, 0.05 ml ofelution buffer (10 mM tris/HCl, pH 8.5) are placed on thechromatographic column and centrifuged through.

EXAMPLE 11

Purification of DNA Fragments from Polyacrylamide (PAA) Gels

The DNA fragment to be isolated is cut out of the PAA gel andtransferred to a 2 ml Eppendorf vessel, the gel is crushed and mixedwith 500 μl of PAA elution buffer (500 mM NH₄Ac, 100 mM MgAc₂, 1 mMEDTA, 0.1% SDS). The mixture is incubated for 30 min at 50° C. and then,after addition of 300 μl of QX1 buffer, is centrifuged through acentrifuge chromatographic column. Then, the centrifuge chromatographiccolumn is washed salt-free with 80% ethanol/water and subsequently iscentrifuged for 1 min to completely remove excess ethanol. For elution,0.1 ml of elution buffer (10 mM tris/HCl, pH 8.5) are placed on thechromatographic column and centrifuged through.

EXAMPLE 12

Purification of Large (>3,000 bp) PCR Fragments

A 100 μl PCR amplification reaction is mixed with 500 μl of QXB buffer(5 M GuHCl); preliminary separation of the paraffin oil layer coveringthe reaction mixture is not necessary. Further purification is performedas in example 4.

EXAMPLE 13

Purification of Single-stranded PCR Products

A 100 μl amplification mixture of an asymmetrical PCR is mixed with 500μl of PB buffer (5 M GuHCl, 30% isopropanol). Further purification isperformed as in example 4. For elution, 0.05 ml of elution buffer (10 mMtris/HCl, pH 8.5) are placed on the chromatographic column andcentrifuged through. The eluate will contain about 90% of thesingle-stranded PCR product which may directly be used for the secondamplification of for sequencing.

EXAMPLE 14

Total Nucleic Acids Preparation from Tissues or Cells

Up to 50 mg of a tissue or up to 10⁶ cells are homogenized in 400 μl ofa buffered chaotropic solution (4 M GTC, 25 mM sodium citrate, pH 7.5,2% 2-mercaptoethanol), optionally mixed with a 5-100% detergent (NP40,Tween 20, Triton-X-100, SDS, CTAB, Sarkosyl). In this step, efficientlysis of all eukaryotic and/or prokaryotic cells and/or viruses (withconcomitant inactivation of infective pathogens) and denaturing ofproteins (especially ribonucleases; with concomitant removal of theproteins bound to the nucleic acids) are taking place simultaneously.Adding 260 μl of a 100% alcohol (methanol, ethanol, n-propanol,isopropanol) provides highly specific binding conditions for nucleicacids. The lysate thus adjusted is transferred to the device.

Subsequently, impurities such as proteins, heme, heparin, metabolitesand polysaccharides are washed out with 700 μl of a washing bufferconsisting of 1 M GTC, 25 mM tris/HCl, pH 7.5, 400% of an alcohol(methanol, ethanol, n-propanol, isopropanol) and with 700 μl of awashing buffer consisting of 10 mM tris/HCl, pH 7.5, 80% of an alcohol(methanol, ethanol, n-propanol, isopropanol). The nucleic acids areeluted either with a buffer with low concentration of salts (10 mM tris,pH 7.5) or with destined (deionized) water.

What is claimed is:
 1. A method for the purification and separation of anucleic acid mixture by chromatography, comprising the steps of: a)adsorbing on a substrate the nucleic acid mixture from an aqueousadsorption solution containing (i) salts effecting a high ionic strengthand (ii) 1 to 50% by volume of at least one C₁-C₅ aliphatic alcohol orpolyethylene glycol or at least one C₁-C₅ aliphatic alcohol andpolyethylene glycol wherein said substrate comprises a porous ornon-porous mineral substrate selected from the group consisting ofsilica gel, glass fibers, quartz fibers, and zeolites, followed by b)optionally washing said substrate with a washing solution; followed byc) eluting said nucleic acid mixture with a solution having a lowerionic strength than the aqueous adsorption solution, effecting thereby anucleic-acid fraction; and d) collecting the nucleic-acid fraction. 2.The method according to claim 1, wherein the salts in the adsorptionsolution are chaotropic salts in concentrations of from 1 to 8 M.
 3. Themethod of claim 2, wherein the claotropic salts are selected from thegroup consisting of sodium perchlorate, guanidine hydrochloride,guanidine isothiocyanate, and sodium iodine.
 4. The method according toclaim 1, wherein the salts are present in the adsorption solution at aconcentration of 1 to 10 M and are selected from the group consisting oflithium chloride, sodium chloride, potassium chloride, sodium acetate,urea, and mixtures thereof.
 5. The method according to claim 1, whereinthe mineral substrate has a particle size of 0.1 μm to 1,000 μm.
 6. Themethod according to claim 1, wherein the substrate is porous, havingpore sizes of 2 to 1,000 nm.
 7. The method according to claim 1, whereinsaid porous or non-porous substrate is present in the form of loosefillings.
 8. The method according to claim 7, wherein said substrate iscomprised of zeolites.
 9. The method according to claim 1, wherein saidporous or non-porous substrate is present in the form of (i) filterlayers of glass fibers or quartz fibers, (ii) a membrane in which silicagel is incorporated, (iii) fibers of quartz, or (iv) glass wool.
 10. Themethod according to claim 9, wherein said substrate is comprised ofzeolites.
 11. The method according to claim 1, wherein said nucleicacids to be separated and purified are derived from cell cultures,tissues, body fluids, feces, or microorganisms.
 12. The method accordingto claim 11, wherein said nucleic acids to be separated and purified arederived from bacteria or viruses.
 13. The method according to claim 11,wherein said nucleic acids to be separated and purified are derived frommycobacterium tuberculosis, cytomegalo-virus, HIV, hepatitis B,hepatitis C, or hepatitis viruses.
 14. The method according to claim 11,wherein said nucleic acids to be separated and purified are obtained bypolymnerase chain reaction (PCR).
 15. The method according to claim 11,wherein said nucleic acids to be separated and purified are plasmid DNA,genomic DNA, or RNA.
 16. The method according to claim 11, wherein saidnucleic acids to be separated and purified are plasmid DNA, chromosomal,DNA, or RNA from microorganisms.
 17. The method according to claim 11,wherein said nucleic acids to be separated and purified are obtainedfrom sequence analyses.
 18. The method according to claim 1, whereinafter fractionating less than 10% of said nucleic acids are shorter than10 kb.
 19. The method according to claim 1, wherein said nucleic acidsare oligonucleotides.
 20. The method according to claim 1, wherein thesubstrate is a membrane of silica gel, and the method further comprises,before the adsorbing step, the steps of: lysing a cell sample containingthe nucleic acids; followed by adjusting conditions for adsorption onthe silica gel in one single step.
 21. The method according to claim 1,wherein said nucleic acids are plasmids, and lysing the cell sample isby alkaline lysis.
 22. The method according to claim 21, wherein theadsorbing step is performed in the presence of detergents.
 23. Themethod according to claim 1, further comprising, after said collectingstep, file step of modifying the nucleic acids.